Dual-function chromogenic screening-based CRISPR/Cas9 genome editing system for actinomycetes

Qiushui Wang, Feng Xie, Yaojun Tong, Rebecca Habisch, Bowen Yang, Lixin Zhang, Rolf Müller, Chengzhang Fu*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Actinobacteria are one of the most important sources of pharmaceutically valuable and industrially relevant secondary metabolites. Modern genome mining reveals that the potential for secondary metabolite production of actinomycetes has been underestimated. Recently, the establishment of CRISPR/Cas9-based genetic manipulation approaches in actinomycetes opened a new era for genome engineering of this type of organism. Compared with the traditional methods, the application of CRISPR/Cas9 shows several advantages in actinomycetes including higher efficiency and ease of operation. However, the screening process for the correctly edited mutants and the plasmid curing are still time- and labor-intensive. To address this problem, we developed an updated version of the CRISPR/Cas9 genome editing system for actinomycetes, based on two chromogenic reporter systems (GusA and IdgS). Our system facilitates both processes of positive clone screening and plasmid curing. Here, we demonstrate by three case studies in both model actinomycetes and non-model actinomycetes that this system is faster and more efficient. We performed the deletion of one single gene, actIORFI (SCO5087 of the actinorhodin gene cluster) in Streptomyces coelicolor M145, one small-size (5.5 kb) gene cluster (orange-pigmented carotenoid gene cluster), and one relatively large-size (61 kb) gene cluster (abyssomicin gene cluster) in Verrucosispora sp. MS100137. The results presented in this study indicate that this updated CRISPR/Cas9 system employing chromogenic reporters is versatile and broadly applicable in genome engineering of actinomycetes, not only for the largest genus Streptomyces.
Original languageEnglish
JournalApplied Microbiology and Biotechnology
Volume104
Pages (from-to)225-239
ISSN0175-7598
DOIs
Publication statusPublished - 2020

Cite this

Wang, Qiushui ; Xie, Feng ; Tong, Yaojun ; Habisch, Rebecca ; Yang, Bowen ; Zhang, Lixin ; Müller, Rolf ; Fu, Chengzhang. / Dual-function chromogenic screening-based CRISPR/Cas9 genome editing system for actinomycetes. In: Applied Microbiology and Biotechnology. 2020 ; Vol. 104. pp. 225-239.
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title = "Dual-function chromogenic screening-based CRISPR/Cas9 genome editing system for actinomycetes",
abstract = "Actinobacteria are one of the most important sources of pharmaceutically valuable and industrially relevant secondary metabolites. Modern genome mining reveals that the potential for secondary metabolite production of actinomycetes has been underestimated. Recently, the establishment of CRISPR/Cas9-based genetic manipulation approaches in actinomycetes opened a new era for genome engineering of this type of organism. Compared with the traditional methods, the application of CRISPR/Cas9 shows several advantages in actinomycetes including higher efficiency and ease of operation. However, the screening process for the correctly edited mutants and the plasmid curing are still time- and labor-intensive. To address this problem, we developed an updated version of the CRISPR/Cas9 genome editing system for actinomycetes, based on two chromogenic reporter systems (GusA and IdgS). Our system facilitates both processes of positive clone screening and plasmid curing. Here, we demonstrate by three case studies in both model actinomycetes and non-model actinomycetes that this system is faster and more efficient. We performed the deletion of one single gene, actIORFI (SCO5087 of the actinorhodin gene cluster) in Streptomyces coelicolor M145, one small-size (5.5 kb) gene cluster (orange-pigmented carotenoid gene cluster), and one relatively large-size (61 kb) gene cluster (abyssomicin gene cluster) in Verrucosispora sp. MS100137. The results presented in this study indicate that this updated CRISPR/Cas9 system employing chromogenic reporters is versatile and broadly applicable in genome engineering of actinomycetes, not only for the largest genus Streptomyces.",
author = "Qiushui Wang and Feng Xie and Yaojun Tong and Rebecca Habisch and Bowen Yang and Lixin Zhang and Rolf M{\"u}ller and Chengzhang Fu",
year = "2020",
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Dual-function chromogenic screening-based CRISPR/Cas9 genome editing system for actinomycetes. / Wang, Qiushui; Xie, Feng; Tong, Yaojun; Habisch, Rebecca; Yang, Bowen; Zhang, Lixin; Müller, Rolf; Fu, Chengzhang.

In: Applied Microbiology and Biotechnology, Vol. 104, 2020, p. 225-239.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Dual-function chromogenic screening-based CRISPR/Cas9 genome editing system for actinomycetes

AU - Wang, Qiushui

AU - Xie, Feng

AU - Tong, Yaojun

AU - Habisch, Rebecca

AU - Yang, Bowen

AU - Zhang, Lixin

AU - Müller, Rolf

AU - Fu, Chengzhang

PY - 2020

Y1 - 2020

N2 - Actinobacteria are one of the most important sources of pharmaceutically valuable and industrially relevant secondary metabolites. Modern genome mining reveals that the potential for secondary metabolite production of actinomycetes has been underestimated. Recently, the establishment of CRISPR/Cas9-based genetic manipulation approaches in actinomycetes opened a new era for genome engineering of this type of organism. Compared with the traditional methods, the application of CRISPR/Cas9 shows several advantages in actinomycetes including higher efficiency and ease of operation. However, the screening process for the correctly edited mutants and the plasmid curing are still time- and labor-intensive. To address this problem, we developed an updated version of the CRISPR/Cas9 genome editing system for actinomycetes, based on two chromogenic reporter systems (GusA and IdgS). Our system facilitates both processes of positive clone screening and plasmid curing. Here, we demonstrate by three case studies in both model actinomycetes and non-model actinomycetes that this system is faster and more efficient. We performed the deletion of one single gene, actIORFI (SCO5087 of the actinorhodin gene cluster) in Streptomyces coelicolor M145, one small-size (5.5 kb) gene cluster (orange-pigmented carotenoid gene cluster), and one relatively large-size (61 kb) gene cluster (abyssomicin gene cluster) in Verrucosispora sp. MS100137. The results presented in this study indicate that this updated CRISPR/Cas9 system employing chromogenic reporters is versatile and broadly applicable in genome engineering of actinomycetes, not only for the largest genus Streptomyces.

AB - Actinobacteria are one of the most important sources of pharmaceutically valuable and industrially relevant secondary metabolites. Modern genome mining reveals that the potential for secondary metabolite production of actinomycetes has been underestimated. Recently, the establishment of CRISPR/Cas9-based genetic manipulation approaches in actinomycetes opened a new era for genome engineering of this type of organism. Compared with the traditional methods, the application of CRISPR/Cas9 shows several advantages in actinomycetes including higher efficiency and ease of operation. However, the screening process for the correctly edited mutants and the plasmid curing are still time- and labor-intensive. To address this problem, we developed an updated version of the CRISPR/Cas9 genome editing system for actinomycetes, based on two chromogenic reporter systems (GusA and IdgS). Our system facilitates both processes of positive clone screening and plasmid curing. Here, we demonstrate by three case studies in both model actinomycetes and non-model actinomycetes that this system is faster and more efficient. We performed the deletion of one single gene, actIORFI (SCO5087 of the actinorhodin gene cluster) in Streptomyces coelicolor M145, one small-size (5.5 kb) gene cluster (orange-pigmented carotenoid gene cluster), and one relatively large-size (61 kb) gene cluster (abyssomicin gene cluster) in Verrucosispora sp. MS100137. The results presented in this study indicate that this updated CRISPR/Cas9 system employing chromogenic reporters is versatile and broadly applicable in genome engineering of actinomycetes, not only for the largest genus Streptomyces.

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DO - 10.1007/s00253-019-10223-4

M3 - Journal article

C2 - 31788711

VL - 104

SP - 225

EP - 239

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

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